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cancers

Review
Exploring Predictive and Prognostic Biomarkers in Colorectal
Cancer: A Comprehensive Review
Karam Ashouri 1,† , Alexandra Wong 1,† , Pooja Mittal 1 , Lesly Torres-Gonzalez 1 , Jae Ho Lo 1 , Shivani Soni 1 ,
Sandra Algaze 1 , Taline Khoukaz 1 , Wu Zhang 1 , Yan Yang 1 , Joshua Millstein 2 , Heinz-Josef Lenz 1, *,‡
and Francesca Battaglin 1, *,‡

1 Division of Medical Oncology, Norris Comprehensive Cancer Center, Keck School of Medicine,
University of Southern California, Los Angeles, CA 90089, USA; [email protected] (K.A.);
[email protected] (A.W.)
2 Department of Population and Public Health Sciences, Norris Comprehensive Cancer Center,
Keck School of Medicine, University of Southern California, Los Angeles, CA 90089, USA
* Correspondence: [email protected] (H.-J.L.); [email protected] (F.B.);
Tel.: +1-(323)-865-3967 (H.-J.L.); +1-(323)-865-3930 (F.B.)
† These authors contributed equally to this work as co-first authors.
‡ These authors contributed equally to this work as co-last authors.

Simple Summary: Colorectal cancer is a major health concern globally, and finding ways to improve
treatment outcomes is crucial. This review explores the role of biomarkers—biological indicators that
can predict how a patient will respond to treatment or indicate the likely course of the disease—in
managing colorectal cancer. By examining both well-established and emerging biomarkers, we hope
to provide a clearer understanding of how these markers can guide personalized treatment plans. The
findings from this research could help doctors make more informed decisions, ultimately improving
patient care and outcomes in colorectal cancer.

Abstract: Colorectal cancer (CRC) remains the second leading cause of cancer-related mortality
worldwide. While immune checkpoint inhibitors have significantly improved patient outcomes,
Citation: Ashouri, K.; Wong, A.; their effectiveness is mostly limited to tumors with microsatellite instability (MSI-H/dMMR) or
Mittal, P.; Torres-Gonzalez, L.; Lo, J.H.; an increased tumor mutational burden, which comprise 10% of cases. Advancing personalized
Soni, S.; Algaze, S.; Khoukaz, T.;
medicine in CRC hinges on identifying predictive biomarkers to guide treatment decisions. This
Zhang, W.; Yang, Y.; et al. Exploring
comprehensive review examines established tissue markers such as KRAS and HER2, highlighting
Predictive and Prognostic Biomarkers
their roles in resistance to anti-EGFR agents and discussing advances in targeted therapies for these
in Colorectal Cancer: A
markers. Additionally, this review summarizes encouraging data on promising therapeutic targets
Comprehensive Review. Cancers 2024,
16, 2796. https://doi.org/10.3390/
and highlights the clinical utility of liquid biopsies. By synthesizing current evidence and identifying
cancers16162796 knowledge gaps, this review provides clinicians and researchers with a contemporary understanding
of the biomarker landscape in CRC. Finally, the review examines future directions and challenges
Academic Editor: Tianhui Hu
in translating promising biomarkers into clinical practice, with the goal of enhancing personalized
Received: 28 June 2024 medicine approaches for colorectal cancer patients.
Revised: 4 August 2024
Accepted: 7 August 2024 Keywords: colorectal cancer; gene expression profiling; molecular targeted therapy; tumor biomarkers
Published: 8 August 2024

1. Introduction
Copyright: © 2024 by the authors.
Licensee MDPI, Basel, Switzerland.
Colorectal cancer (CRC) is the third most common cancer and the second leading cause
This article is an open access article
of cancer-related deaths worldwide [1]. Approximately 20% of patients have metastatic
distributed under the terms and disease upon presentation, and the 5-year survival rate for these patients remains less than
conditions of the Creative Commons 20%, highlighting the need to expand the therapeutic armamentarium in this setting [2].
Attribution (CC BY) license (https:// Over the last two decades, the treatment landscape for metastatic CRC (mCRC) has
creativecommons.org/licenses/by/ significantly evolved. The standard of treatment was 5-fluorouracil (5-FU) combined with
4.0/). leucovorin until it was found that combining 5-FU with oxaliplatin or irinotecan improved

Cancers 2024, 16, 2796. https://doi.org/10.3390/cancers16162796 https://www.mdpi.com/journal/cancers


Cancers 2024, 16, 2796 2 of 25

median overall survival (OS) to 20 months [3]. The advent of immunotherapy has also led to
substantial therapeutic benefits, with a 4-year OS > 70% in mCRC positive for microsatellite
instability or mismatch repair deficiency (MSI-H/dMMR) [4]. However, this biomarker
applies to 4% of all mCRC cases, leaving most patients reliant on traditional chemotherapy
and incremental gains from new biologic agents.
The identification of predictive and prognostic biomarkers with immunohistochem-
istry (IHC) or next-generation sequencing (NGS) is crucial for advancing personalized
medicine in CRC. Predictive biomarkers help determine the likelihood of the response to
specific therapies, while prognostic biomarkers provide information on the overall dis-
ease outcome, regardless of treatment. Notably, CRC primary tumors and metastatic sites
harbor a high concordance (>90%) in their mutational profile for actionable genes [5,6].
Integrating these genetic markers in the four distinct consensus molecular subtypes (CMSs)
has enriched our understanding of the clinical and biological characteristics of CRC [7].
Despite the progress in molecular diagnostics, challenges remain in translating these
biomarkers into clinical practice. The heterogeneity of CRC, both morphologically and
molecularly, complicates the identification of universally applicable biomarkers. The
clinical relevance of many emerging biomarkers is still under investigation, necessitating
further research and validation in larger patient cohorts. This review aims to provide a
comprehensive overview of key established, promising, and potential biomarkers in CRC.

2. Established Biomarkers
2.1. RAS Status
Proteins of the RAS family are GTPases involved in the RAS/RAF/MEK/ERK path-
way, playing a crucial role in cell division and proliferation. RAS mutations are prevalent
across various cancers, making them one of the most common oncogenes. In CRC, RAS
mutations are found in approximately 55% of cases, with KRAS mutations occurring in
about 50%, NRAS in 4%, and HRAS in less than 1% [8].
Most KRAS mutations involve glycine substitutions at positions 12 or 13, leading
to constitutive activation of the GTPase side chain. The most common KRAS mutations
in CRC are KRAS G12D (36%), KRAS G12V (21.8%), and KRAS G13D (18.8%), while
KRAS G12C mutations are found in 3–4% of CRC cases (Figure 1) [9,10]. KRAS muta-
tions are more prevalent in left-sided tumors than right-sided tumors and similarly more
common in microsatellite stable (MSS) CRC relative to MSI-H/dMMR CRC. In frontline
chemotherapy–treated mCRC, the KRAS wildtype status conferred an improved OS relative
to mutated tumors (20.9 months vs. 16.9 months, HR = 0.71, 95% CI 0.57, 0.88, HORIZON II,
NCT00399035) [11]. The prognostic utility of KRAS mutations extends to both young-onset
and late-onset CRC, revealing worse cause-specific survival relative to KRAS wildtype
tumors in both age groups [12]. Among specific KRAS mutations, G12C demonstrated the
worst prognosis relative to wildtype tumors (4.3 months vs. 23.3 months, p < 0.001) [13].
RAS mutations result in constitutive activation downstream of EGFR blockade, ren-
dering epidermal growth factor receptor (EGFR) inhibitors ineffective. Multiple studies, in-
cluding the phase 3 CRYSTAL and PRIME trials, have demonstrated that KRAS/NRAS mu-
tations predict a poor response to combination chemotherapy with EGFR inhibitors [14,15].
Consequently, testing for KRAS/NRAS mutations is recommended by the National Com-
prehensive Cancer Network (NCCN) for all patients with mCRC, because those with these
mutations should not receive panitumumab or cetuximab in combination with chemother-
apy or as monotherapy [16]. Within 12 months of treatment, most CRCs acquire resistance
to anti-EGFR therapies, frequently (~50%) due to secondary KRAS alterations [17,18]. This
underscores the importance of checking for these mutations during anti-EGFR therapy.
ers 2024, 16, x FOR PEER REVIEW 3 o
Cancers 2024, 16, 2796 3 of 25

FigureFigure
1. Current predictive biomarkers for metastatic colorectal cancer (CRC). EGFRi = EGFR i
1. Current predictive biomarkers for metastatic colorectal cancer (CRC). EGFRi = EGFR
bition;inhibition;
VEGFi =VEGFi
VEGF= inhibition; G12Ci
VEGF inhibition; = KRAS
G12Ci = KRASG12C
G12C inhibition; BRAFi
inhibition; BRAFi = BRAF
= BRAF inhibition; I
inhibition;
Immune checkpoint inhibition; HER2i = HER2 inhibition; CRC = colorectal cancer. (Created
ICI = Immune checkpoint inhibition; HER2i = HER2 inhibition; CRC = colorectal cancer. (Created in in
render.com).
Biorender.com).

When selecting biologic agents (bevacizumab, cetuximab, and panitumumab) to


RAS mutations result in constitutive activation downstream of EGFR blockade, r
pair with frontline chemotherapy in mCRC, several studies highlight the superiority of
dering epidermal
anti-EGFR growth
therapies factor and
(cetuximab receptor (EGFR)ininhibitors
panitumumab) ineffective. Multiple
RAS wildtype/left-sided tumors stud
including the
(Figure 1). phase 3 CRYSTAL
The PARADIGM Studyand PRIME trials,
demonstrated improved have demonstrated
OS with panitumumabthat KRAS/NR
versus
mutations predict a poor response to combination chemotherapy with EGFR inhibi
bevacizumab in combination with chemotherapy for first-line treatment in RAS wildtype
patients (37.9 months vs. 34.3 months), with a more pronounced effect in left-sided tu-
[14,15]. Consequently, testing for KRAS/NRAS mutations is recommended by the
tional Comprehensive Cancer Network (NCCN) for all patients with mCRC, beca
those with these mutations should not receive panitumumab or cetuximab in combina
with chemotherapy or as monotherapy [16]. Within 12 months of treatment, most CR
acquire resistance to anti-EGFR therapies, frequently (~50%) due to secondary KRAS
Cancers 2024, 16, 2796 4 of 25

mors [19]. Updated results from the RAS wildtype cohort in the FIRE-3 trial indicated
improved OS with the FOLFIRI plus cetuximab group compared to the FOLFIRI plus beva-
cizumab group (33.1 months vs. 25.0 months; HR = 0.70; p = 0.0059) [20]. The cetuximab
group also demonstrated a more frequent early tumor shrinkage and a greater median
depth of response [20].
Recent advances have led to the development of oral KRAS G12C small molecule
inhibitors. Initial studies of sotorasib and adagrasib monotherapy showed modest effects
in chemo-refractory mCRC, and combining them with EGFR inhibitors appears to en-
hance their efficacy and overcome resistance mechanisms [21]. The KRYSTAL-1 trial, a
phase I/II study examining adagrasib with or without cetuximab, showed a numerically
higher median duration of response and response rates with the addition of cetuximab [22]
(NCT03785249). Soon after, adagrasib was awarded accelerated Food and Drug Administra-
tion (FDA) approval as a second-line agent in combination with cetuximab for KRAS G12C–
mutated CRC. The phase 3 CodeBreak 300 trial evaluates sotorasib in chemo-refractory
mCRC at two doses (240 mg and 960 mg) in combination with panitumumab compared
to a standard of care (SOC) group (Lonsurf or regorafenib) [23]. Early data demonstrate
superior progression free survival (PFS) in both sotorasib groups relative to SOC (960 mg:
5.6 months vs. 240 mg: 3.9 months vs. SOC: 2.2 months) (NCT05198934) [23]. Another
KRAS G12C inhibitor, divarasib, is being studied in a Phase 1 trial as monotherapy and
in combination with cetuximab (NCT04449874) (Table 1). Additional phase 3 studies will
examine adagrasib combined with cetuximab compared to chemotherapy in the second-line
setting (NCT04793958) and sotorasib in combination with panitumumab and chemotherapy
in treatment-naive patients (NCT06252649). Preclinical data suggest that SHP2-inhibitors
augment KRAS G12C inhibitors by improving engagement and downregulating resis-
tance mechanisms, leading to clinical trials evaluating this combination (NCT04330664,
NCT04916236, NCT05288205, NCT04699188, NCT04449874) (Table 1) [24]. In preclinical
CRC models, KRAS mutations increased the expression of amino acid transporters via
the hippo pathway effector YAP1, resulting in mTOR activation and subsequent CRC cell
proliferation [25]. Mechanistic studies in lung cancer indicate that the hippo pathway is
implicated in KRAS G12C resistance, highlighting its therapeutic potential in augmenting
KRAS inhibitors [26]. G12D is the most prevalent KRAS alteration in CRC, with several
small molecular inhibitors (MRTX1133 and RMC-9805) undergoing early phase evaluation
(NCT05737706, NCT06040541) (Table 1). MRTX1133 (G12D inhibitor) has shown synergism
alongside 5-FU in preclinical models, with similar effects in KRAS G12V–mutated cell
lines [27]. Alternative strategies targeting specific KRAS mutations utilize T cell receptors
(TCRs), which recognize intracellular antigens presented by human leukocyte antigen
(HLA) molecules to trigger an immune response [28]. Several clinical trials utilize TCRs
in KRAS G12V (NCT06105021, NCT06043713) and G12D (NCT03948763) CRC (Table 1).
In addition to the mutation selective inhibitors, clinical trials are evaluating pan-KRAS
inhibitors (NCT04975265) (Table 1). RMC-6236 is a pan-RAS inhibitor that targets the full
range of RAS alterations (KRAS, HRAS, NRAS) with activity against mutant and wildtype
RAS variants and is undergoing Phase 1 studies (NCT05379985) (Table 1) [29].
Cancers 2024, 16, 2796 5 of 25

Table 1. Clinical trials evaluating therapeutic potential in KRAS mutations and promising biomarkers.

Gene Molecular Criteria Setting Therapy of Interest Trial Number/Phase Status


mCRC Sotorasib (G12C inhibitor) + panitumumab NCT05198934 (Phase 3) Active
mCRC Adagrasib (G12C inhibitor) + cetuximab NCT03785249 (Phase 1/2) Active
mCRC Adagrasib (G12C inhibitor) + cetuximab NCT04793958 (Phase 3) Active

mCRC Sotorasib (G12C inhibitor) + panitumumab + NCT06252649 (Phase 3) Not yet recruiting
FOLFIRI
KRAS G12C mutation
mCRC Divarasib (G12C inhibitor) + cetuximab NCT04449874 (Phase 1) Active
Glecirasib (G12C inhibitor) + JAB-3312 Active
Advanced solid tumors (SHP2 inhibitor) NCT05288205 (Phase 1/2)

Advanced solid tumors Opnurasib (G12C inhibitor) NCT04699188 (Phase 1/2) Active
Advanced solid tumors Divarasib (G12C inhibitor) NCT04449874 (Phase 1) Active
KRAS KRAS mutated (except Vociprotafib
mCRC NCT04916236 (Phase 1) Active
G13 mutations) (SHP2 inhibitor) + Temuterkib (ERK ½ inhibitor)
Adagrasib (G12C inhibitor) + Batoprotafib
mCRC NCT04330664 (Phase 1/2) Active
(SHP2 inhibitor)
KRAS G12D mutation Advanced solid tumors MRTX1133 (G12D inhibitor) NCT05737706 (Phase 1/2) Active
Advanced solid tumors RMC-9805 (G12D inhibitor) NCT06040541 (Phase 1) Active
Advanced solid tumors AFNT-211 (G12V TCR) NCT06105021 (Phase 1/2) Active
KRAS G12V mutation
Advanced solid tumors FHA11 (G12V TCR) NCT06043713 (Phase 1) Active
KRAS G12D, G12V, G13D or
G12C Non-MSI-H/dMMR mCRC mRNA-5671/V941 NCT03948763 (Phase 1) Completed (2022)

RAS mutation Advanced solid tumors RMC-6236 (RASmulti inhibitor) NCT05379985 (Phase 1) Active
CEACAM5 Advanced solid tumors M9140 (CEACAM5 ADC) NCT05464030 (Phase 1) Active
Obinutuzumab (CD20 mAb) + Cibisatamab
High CEACAM5 Expression Previously treated MSS mCRC (CEA-CD3 bispecific Ab) + atezolizumab NCT03866239 (Phase 1) Completed (2024)
(PDL1 mAb)
c-MET Advanced solid tumors ABBV-400 (MET Ab) + Bevacizumab NCT05029882 (Phase 1) Active
Previously treated unresectable ABBV-400 (MET Ab) + 5FU + Folinic acid + NCT06107413 (Phase 2) Active
mCRC Bevacizumab

PIK3CA mutation Advanced solid cancers including Serabelisib (PI3Kα inhibitor) NCT05300048 (Phase 1b) Active
PIK3CA CRC
PIK3CA mutation mCRC Alpelisib (PI3Kα inhibitor) + capecitabine NCT04753203 (Phase 1b/2) Active
Cancers 2024, 16, 2796 6 of 25

Table 1. Cont.

Gene Molecular Criteria Setting Therapy of Interest Trial Number/Phase Status

CXCR4 Advanced pancreatic, ovarian, and


CRC Plerixafor (CXCR4 inhibitor) NCT02179970 (Phase 1) Completed 2018

mCRC Maraviroc (CCR5 inhibitor) NCT01736813 (phase 1) Completed 2014


Advanced solid cancers including OB-002 (CCR5 antagonist) NCT05940844 (Phase 1) Not yet recruiting
CRC
CCR5 MSS mCRC Vicriviroc (CCR5 inhibitor) + pembrolizumab NCT03631407 (Phase 2) Completed 2021
Ipilimumab + Maraviroc (CCR5 inhibitor) +
Advanced pancreatic and CRC NCT04721301 (Phase 1) Completed 2023
nivolumab
MSS mCRC Maraviroc (CCR5 inhibitor) + pembrolizumab NCT03274804 (Phase 1) Completed 2020
None for inclusion but
TIM3 evaluating TIM3 expression Advanced solid cancers including TSR-022 (anti-TIM3) + multiple ICI/chemotherapy NCT02817633 (Phase 1) Active
CRC arms
levels
Advanced solid cancers including TSR-033 (LAG3 antibody) + dostarlimab NCT03250832 (Phase 1) Completed 2023
CRC
MSI-H Localized and locally advanced CRC Fianlimab (LAG3 inhibitor) + Cemiplimab NCT06205836 (Phase 1) Active
MSS, cohort A CPM ≥ 15%, Metastatic or locally advanced CRC Relatlimab (LAG3 antibody) + Nivolumab NCT03642067 (Phase 2) Active
LAG3 cohort B CPM < 15%.
Advanced solid cancers including XmAb® 22841 (CTLA-4 × LAG3 bispecific antibody) NCT03849469 (Phase 1) Completed 2023
CRC + Pembrolizumab
MSS CPS ≥ 1 mCRC Favezelimab (LAG3 antibody) + Pembrolizumab NCT05064059 (Phase 3) Active
Tislelizumab (PD-1 Antibody) + Fruquintinib
ARID1A MSS and ARID1A mutation mCRC NCT05690035 (Phase 2) Active
(VEGFR 1/2/3 Inhibitor)
KRAS mutated and mCRC Onvansertib (PLK1 inhibitor) + FOLFIRI + NCT03829410 (Phase 1b/2) Completed 2024
BRAFV600E negative Bevacizumab
KRAS mutated and mCRC Onvansertib (PLK1 inhibitor) + Bevacizumab + NCT06106308 (Phase 2) Active
PLK1 BRAFV600E negative FOLFIRI or FOLFOX

KRAS or NRAS Mutation mCRC Onvansertib (PLK1 inhibitor) + Bevacizumab + NCT05593328 (Phase 2) Active
FOLFIRI
CRC = colorectal cancer; mCRC = metastatic colorectal cancer; MSS = microsatellite stable; CPM = composite PD-L1/Mucin; CPS = combined positive score.
Cancers 2024, 16, 2796 7 of 25

2.2. BRAF Mutations


The RAF family of serine/threonine kinases are components of the RAS/RAF/MAPK/ERK
signaling pathway and contain the following three members: ARAF, BRAF, and CRAF [30].
Under physiological conditions, RAF proteins exist as monomers within the cytosol un-
til they are recruited to the membrane, bind activated RAS, and form active homo- or
hetero-dimers [31]. Once activated, RAF dimers go on to activate MEK1/2, subsequently
activating the ERK1/2 transcription complex, which regulates cell proliferation, differentia-
tion, survival, senescence, and apoptosis [30–32]. The most common BRAF mutation results
in valine substitution for glutamic acid at amino acid 600 (BRAFV600E ), stimulating the
MEK/ERK pathway in tumor cells (Figure 1) [33,34]. Clinical and pathological phenotypes
associated with BRAFV600E -mutated CRC include proximal tumor location, aggressive
growth, unfavorable metastasizing patterns, EGFR blockade resistance, and reduced over-
all survival (Figure 1) [35]. Pooled analysis from three mCRC clinical trials (COIN, FOCUS,
PICCOLO) noted an increased frequency of MSI-H/dMMR with BRAF mutations com-
pared to the BRAF wildtype (12.6% vs. 3.0%, p < 0.001) [36]. When combining the two
frontline trials (COIN, FOCUS), BRAF mutant tumors suffered from worse OS (10.8 months
vs. 16.4 months, HR = 1.49, p < 0.001) and remained significant when adjusting for con-
founders [36]. The negative prognostic impact of BRAF mutations has been confirmed in
several other studies [37–40]. Non-V600E BRAF mutations occur less frequently in mCRC
(2.2% vs. 7.8%) but offer better prognoses than those with the V600E mutation (p < 0.001)
(Figure 1) [41]. Several retrospective studies and meta-analyses identified BRAF V600E as a
negative predictor for a response to anti-EGFR therapy [42–44]. However, the predictive
value of BRAF non-V600E mutations remains unclear (Figure 1) [45,46].
Therapies that target mutated BRAF include the SOC in melanoma, non-small cell
lung cancer, and some thyroid malignancies. However, monotherapy with vemurafenib or
dabrafenib is ineffective in V600E mCRC due to alternative MAPK activation mechanisms
maintaining elevated MEK activity [35,47]. Co-inhibition of BRAF and the alternative
activation mechanisms targeting EGFR or MEK have demonstrated successful reduction in
MAPK signaling activity and improved patient treatment response [48–50]. Compared to
chemotherapy control (FOLFIRI or irinotecan + cetuximab) in pretreated BRAFV600E mCRC,
both doublet (encorafenib + cetuximab) and triplet therapy (encorafenib + binimetinib
+ cetuximab) demonstrated superior OS (5.9 months vs. 9.3 months vs. 9.3 months,
respectively) and PFS (1.5 months vs. 4.3 months vs. 4.5 months, respectively) in the phase
III BEACON trial [51]. Given that adverse events were more frequent with triplet therapy
but had similar efficacy, the encorafenib plus cetuximab combination is an approved second-
line regimen in BRAFV600E mCRC, with ongoing trials evaluating it in the first-line setting
(Figure 1) (phase III BREAKWATER trial NCT04607421).
Notably, preclinical models in microsatellite stable (MSS) CRC demonstrate that com-
bined BRAF/EGFR inhibition induces a transient MSI-H/dMMR phenotype [52]. En-
corafenib, cetuximab, and nivolumab combination therapy in MSS/BRAFV600E recently
demonstrated encouraging PFS (7.4 months) and OS (15.1 months) with a randomized
phase II trial underway [53] (NCT05308446).

2.3. HER2
HER2, encoded by ERBB2, is an EGFR tyrosine kinase family member. Its overex-
pression and/or amplification have been reported in various solid tumors, particularly
breast cancer, where HER2-targeted therapies have been highly successful. The NCCN
Guidelines now recommend HER2 testing for all mCRC patients, present in 2–6% of cases
given therapeutic implications with anti-EGFR and HER2 directed therapy (Figure 1).
HER2 is measured by IHC, fluorescence in situ hybridization (FISH), or NGS. HER2 over-
expression is indicated by an IHC score of 3+, while an IHC score of 2+ is equivocal and
should prompt FISH testing. A FISH ratio ≥2 confirms HER2 amplification [54]. HER2 am-
plification is more common in left-sided tumors and is more prevalent in RAS/BRAF
wildtype tumors [55,56]. Studies have shown that HER2 overexpression may confer re-
Cancers 2024, 16, 2796 8 of 25

sistance to EGFR inhibition (Figure 1) [55,57,58]. On the other hand, a recent analysis
from the CALGB/SWOG 80405 trial found that high HER2 expressing mCRC (tumor gene
expression above the median) had a longer PFS and OS than tumors with lower HER2
expression [59]. In patients with lower HER2 expression, treatment with cetuximab was
associated with a worse PFS and OS compared to bevacizumab. The poor prognostic
implication of decreased HER2 expression was confirmed in RAS wildtype mCRC based
on the MONSTAR-SCREEN-2 cohort [60]. These findings suggest that HER2 expression
is prognostic and may contribute to selecting anti-EGFR versus anti-VEGF treatment in
the first-line metastatic setting [59]. However, other studies have reported mixed findings
regarding the prognostic role of HER2, and further prospective validation is needed [61].
HER2 is an established therapeutic target in CRC. In the second-line setting for mCRC,
RAS/BRAF wildtype patients with HER2 overexpression/amplification who have not
received prior HER2 inhibitors can be treated with trastuzumab in combination with
pertuzumab, lapatinib, or tucatinib. The HERACLES trial, a phase 2 study of heavily
pre-treated patients with the KRAS wildtype, HER2+ mCRC, demonstrated the efficacy
of dual HER2 blockade using trastuzumab and lapatinib, with an objective response rate
(ORR) of 30% [56]. The MyPathway basket study of trastuzumab and pertuzumab in
treatment-refractory CRC reported an ORR of 38% [62]. The combination of trastuzumab
and tucatinib was the first HER2-directed treatment approved by the FDA for chemo-
refractory mCRC, based on the MOUNTAINEER trial (ORR 38%) [63]. The ongoing phase
3 trial, MOUNTAINEER-03 (NCT05253651), is investigating tucatinib and trastuzumab
in combination with chemotherapy in the first-line setting compared to standard care in
HER2+ patients. In the DESTINY-CRC01 phase 2 trial of patients with HER2+ mCRC who
had received at least two prior lines of therapy, fam-trastuzumab deruxtecan was associated
with an ORR of 45% [64]. Notably, fam-trastuzumab deruxtecan is an option for patients
who received prior HER2-directed therapy; however, the cohort included only patients
with HER2 IHC 3+, and patients must be monitored closely for the potential serious side
effect of interstitial lung disease [64]. Future studies will help elucidate ideal sequencing
strategies for targeted therapy in this group of patients.

2.4. MSI-H
Microsatellites are short tandem repeated sequences spread throughout DNA. Mi-
crosatellite instability is caused by defects in the mismatch repair system, most commonly
due to somatic mutations of MLH1, MSH2, MSH6, PMS2, and EPCAM, which lead to
defects during DNA replication [65,66]. The prevalence of MSI-H/dMMR decreases with
the advancing CRC stage as follows: stage II (20%), then stage III (12%), and stage IV
(4%) (Figure 1) [67]. Knowledge of the MSI-H/dMMR status is critical in managing CRC
for several reasons. Patients with MSI-H/dMMR tumors must be screened for germline
mutations in the mismatch repair genes, which leads to Lynch Syndrome, altering the
treatment of patients and relatives [68]. With the introduction of immune checkpoint
inhibitors (ICI), MSI-H/dMMR status has offered profound survival benefits and cures
for some CRCs [69]. The high neoantigen load of MSI-H/dMMR cancer elicits a strong
antitumor immune response, further augmented with ICIs (Figure 1) [70]. Additionally,
MSI-H/dMMR CRCs have a favorable tumor microenvironment (TME) with increased
tumor-infiltrating lymphocytes and decreased immunosuppressive cells like regulatory T
cells (Tregs) and myeloid-derived suppressor cells (MDSCs) [71,72]. ICIs further enhance
the activity of these preexisting tumor infiltrating lymphocytes (TILs) to target the tumor.
The phase II KEYNOTE-016 trial was the first to demonstrate superior ORR (40%) in
MSI-H/dMMR mCRC relative to MSS (0%) with pembrolizumab [73]. Pembrolizumab’s
superior antitumor activity was confirmed in the KEYNOTE-164 trial, demonstrating a
median PFS and OS of 4.1 and 47.0 months, respectively, in pretreated CRC (chemotherapy
arm PFS: 2.3 months, OS: 31.4 months) [69,74]. The 50% increase in OS relative to the SOC
and 30% PFS plateau at 5 years, all with fewer adverse events in the experimental arm, were
unheard of in previous CRC trials. A similar efficacy was demonstrated with nivolumab
Cancers 2024, 16, 2796 9 of 25

(phase II Checkmate 142 trial), awarding both agents FDA approval for second-line use in
2017 [75]. Phase III KEYNOTE-177 demonstrated that first-line pembrolizumab (200 mg
q3w for up to 2 years) was superior to standard chemotherapy (mFOLFOX6 or FOLFIRI
q2w ± bevacizumab or cetuximab) in MSI-H/dMMR mCRCs, confirming a longer median
PFS (16.5 months vs. 8.2 months, HR 0.60, p = 0.0002) [76]. The effect of nivolumab was
augmented with the addition of anti-CTLA4 (ipilimumab) therapy (24-month PFS: 74%
and OS: 79%) [77]. Phase III Checkmate-8HW confirmed the superior PFS of nivolumab
plus ipilimumab compared to chemotherapy (not reached (NR) vs. 5.9 months; HR 0.21,
p < 0.0001) after 31.5 months of median follow-up in the first-line setting [4].
Despite the clinical benefit, nearly 30% of MSI-H/dMMR tumors are resistant to
ICI [78]. Preclinical data indicate that inhibition of VEGF may promote an immune permis-
sive TME [79,80]. Following encouraging safety and efficacy data, atezolizumab plus beva-
cizumab plus FOLFOX is being evaluated as a first-line regimen in MSI-H/dMMR mCRC
(NCT02997228) [81]. Additional studies combine VEGF inhibitors with pembrolizumab or
nivolumab as second-line agents (NCT05035381).

2.5. Tumor Mutational Burden (TMB)


Tumor mutational burden (TMB) is an established biomarker for predicting the efficacy
and response to ICIs in multiple cancer types, including CRC, independent of the MSI-
H/dMMR status [82]. TMB is an independent biomarker, and studies have shown TMB to
be elevated in MSI-H/dMMR tumors. A high TMB (TMB > 37.4 mutations/megabase [Mb])
stratified patients with a better response to ICIs in an MSI-H/dMMR mCRC cohort [82].
TMB can be expressed as the number of acquired/somatic mutations (coding errors,
base substitutions, and insertions/deletions) per Mb of the sequenced DNA. NGS and
whole exome sequencing (WES) of the tumor genome are used to measure TMB, and
examples of diagnostic NGS panels include MSK-IMPACT (Memorial Sloan Kettering) and
TSO500 (Illumina) [83,84]. A high TMB could potentially translate into higher loads of
neoantigens, stimulating the immune system to recognize and attack tumor cells and thus
increasing sensitivity to immunotherapy [85]. Although only a few somatic mutations may
give rise to neoantigens, even fewer of these are processed by major histocompatibility
complex (MHC) molecules and recognized by T cells as immunogenic. Still, TMB gives a
useful estimation of the tumor neoantigen loads [86]. A recent study by Wang et al. (2022)
reported a high TMB as an indicator of better prognosis in patients with KRAS-mutated
CRC. The study revealed that among CRC patients with KRAS mutations, those with higher
TMB (TMB > 10 mutations per Mb) had a longer OS compared to the ones with lower TMB
(TMB ≤ 10 mutations per Mb) [84].
The US FDA approved the use of pembrolizumab for the treatment of cancer patients
with TMB of >10 mutations per Mb in 2020 (Figure 1) [87]. However, the variation in
intratumoral heterogeneity is one of the factors to be considered while characterizing
TMB across different tumor types. Cancers such as melanoma, non-small cell lung cancer
(NSCLC), CRC, and other squamous carcinomas exhibit a high TMB, while pediatric tumors
and leukemias show the lowest levels of TMB [86]. Due to variation in TMB by cancer type,
more studies are warranted to develop TMB into a robust predictive marker of ICI efficacy.

2.6. NTRK Fusions


The neurotrophic tyrosine receptor kinase (NTRK) genes include NTRK1, NTRK2, and
NTRK3, which encode TRKA, TRKB, and TRKC, respectively. Binding with their respective
ligands leads to the transcription of genes involved in neuronal cell differentiation and
survival mediated by the MAPK, PI3K, and PKC signaling pathways [88]. The alterations in
the NTRK genes have been implicated in the induction of carcinogenesis in both neuronal
and non-neuronal cells. These alterations could be in the form of gene mutations, fusions,
and deletions. The NTRK fusions have been reported in several cancers in both adult and
pediatric populations, with an approximately 80% frequency in rare cancers (including
mammary secretory carcinoma and congenital infantile fibrosarcoma). The frequency of
Cancers 2024, 16, 2796 10 of 25

NRTK fusion is comparably less (commonly <5%) in common cancers, with the frequency
being 0.7–1.5% in CRC (Figure 1) [89].
A recent report by Wang et al. (2022) documented that frequent coalterations in APC
and TP53 genes were found with NTRK fusions, while alterations in RAS/BRAF were
mutually exclusive. NTRK fusion-positive tumors showed a high TMB, MSI-H/dMMR,
and enrichment in POLE/POLD1 mutations compared to molecularly unstratified CRC
tumors. Typically, the NTRK fusion proteins contain the kinase domain of NTRK juxta-
posed with a different gene. In several cases, this leads to the constitutive activation of
downstream signaling pathways such as MAPK and PI3K. NTRK fusions are clinically
targetable mutations, and two first-generation NTRK tyrosine kinase inhibitors (TKIs),
larotrectinib and entrectinib, have been approved by the FDA for the treatment of both
adult and pediatric cancers (Figure 1). Second or next-generation NTRK inhibitors, which
could overcome the resistance to first-generation NTRK TKIs, are also in clinical trials,
including LOXO-195 and TPX-0005 [88,90].
Another study documented the impact of NTRK fusions on outcomes in patients with
locally advanced or metastatic solid cancers. The NTRK fusion-positive cohort (which
included 10 different cancer types with the highest number of patients having CRC) showed
a hazard ratio of 1.6 (p = 0.05) and a median OS of 10.2 months compared to the NTRK
fusion-negative cohort (median OS = 10.4 months). This study suggested that NTRK
fusions might be a negative prognostic factor; however, further studies are warranted [91].

3. Promising Biomarkers
3.1. PD-L1
PD-L1 is expressed at low levels on antigen-presenting cells and a variety of non-
hematopoietic cells, binding PD-1 on T cells [92]. This interaction inhibits T cell activation
and proliferation, suppressing the immune response against tumor cells and allowing
them to evade immune surveillance [93]. In theory, higher PD-L1 expression in tumor
tissue should confer an improved response to ICI treatment. While PD-L1 is a predictive
biomarker in NSCLC, melanoma, and renal cell cancer, its use in CRC is limited [94,95].
PD-L1 expression is induced by IFN-y and TNF-α, secreted by TILs, particularly CD8+ T
cells [92]. Therefore, PD-L1 expression by IHC is highly dependent on spatial heterogeneity
and sampling. Additionally, the plethora of primary antibodies, staining conditions, and
intertumoral heterogeneity add to the technical and biological discrepancy in PD-L1 expres-
sion, limiting its use as a biomarker [96]. Analysis of nivolumab plus ipilimumab–treated
MSI-H/dMMR CRC showed no difference in ORR utilizing PD-L1 ≥ 1% as a cutoff [97].
Meta-analyses have demonstrated positive tumor cell PD-L1 expression to confer worse
OS, but other studies did not find this association or found the opposite effect [98–100].
Some studies have highlighted an increased PD-1 expression in immune cells and TILs to
confer an improved OS in CRC [100,101]. While the utility of PD-1/PD-L1 as a biomarker
requires further research, additional therapies targeting PD-1, such as balstilimab, are being
investigated in colorectal cancer (NCT05608044).

3.2. PI3K
PIK3CA is a key driver gene that encodes a subunit of phosphatidylinositol 3-kinase
(PI3K), a critical component of the PI3K/AKT/mTOR pathway involved in prolifera-
tion, survival, and angiogenesis [102]. PIK3CA mutations activate the PI3K/AKT/mTOR
pathway downstream of RAS activation, conferring resistance to anti-EGFR therapy in
KRAS-wildtype mCRC, with a more pronounced effect in exon 20 alterations [103–105].
The prognostic implication of PIK3CA mutations remains unclear in CRC. Although some
studies have reported a worse prognostic effect with PIK3CA mutations in mCRC, others
have reported a null association [104,106]. Evaluation of serabelisib (PIK3 inhibitor) in solid
tumor patients with PIK3CA mutations is in a phase 1b trial (NCT05300048) (Table 1).
Cancers 2024, 16, 2796 11 of 25

3.3. CXCR4 Axis


CXC chemokine receptor 4 (CXCR4) and its ligand CXCL12 participate in CRC growth,
invasion/metastasis, and angiogenesis [107]. Activation of CXCR4 on tumor cells stimu-
lates ICAM-1, enhancing their adhesion to endothelial cells, while CXCR4 on immune cells
suppresses the intratumoral immune reaction [108–110]. An increased CXCR4 expression
has been linked with an increased liver metastasis and a decreased OS (median 10 months
vs. 27 months) in mCRC [111,112]. Additionally, CXCR4 predicts worse recurrence-free
survival following curative hepatectomy in CRC patients [113]. Preclinical models have
implicated CXCR4 in resistance to oxaliplatin (OXA) and 5-FU [114]. Plerixafor, a small
molecular antagonist of the CXCR4 receptor, is being evaluated in phase 1 studies for
pancreatic and colorectal cancer (NCT02179970) (Table 1).

3.4. CCR5 Axis


The C-C motif chemokine receptor 5 (CCR5)/C-C motif chemokine ligand 5 (CCL5)
axis has been implicated in an autocrine and paracrine fashion to stimulate cancer cell
proliferation, metastasis, and immune regulation [115]. CCR5 induces vascular endothelial
growth factor A (VEGF-A) expression to promote angiogenesis and recruits immunosup-
pressive tumor-associated macrophages (TAMs) and myeloid-derived suppressor cells
(MDSCs) [116]. An increased CCR5 expression showed a favorable treatment interac-
tion toward bevacizumab, while a decreased expression predicted a response to cetux-
imab in the CALGB/SWOG 80405 cohort [117]. Additionally, single nucleotide poly-
morphisms (SNPs) in CCL5/CCR5 have been predictive of a response to cetuximab and
regorafenib therapy [118–120]. CCR5 may have a prognostic effect, as one study noted
that an increased CCR5 expression displayed worse disease-specific survival, and the
CALGB/SWOG 80405 cohort similarly displayed a decreased OS in cetuximab/FOLFOX-
treated mCRC [117,121]. Maraviroc, an FDA-approved HIV medication that inhibits CCR5
expression, showed initial promise in treating refractory mCRC (phase 1 MARACON
trial NCT01736813). Given the immunosuppressive role of CCR5 and their expression on
immune cells, maraviroc and another CCR5 inhibitor, vicriviroc, are being evaluated in
combination with pembrolizumab for MSS mCRC (NCT03274804, NCT03631407) (Table 1).

3.5. TIM3/LAG3
T cell immunoglobulin and mucin domain-containing protein 3 (TIM3) and lymphocyte-
activation gene 3 (LAG3) are immune checkpoint receptors expressed on T cells, macrophages, and
other immune cells, which suppresses antitumor immune responses [122,123]. Within CRC
TME, TIM3 and LAG3 promote immune tolerance by inhibiting T cell activity and favoring
M2-like polarization of macrophages [124,125]. In stage I-III CRC, an increased tumor TIM3
and LAG3 expression correlated with a decreased OS, while an increased expression on
immune cells had the opposite effect [126]. Murine models revealed that the co-blockade
of TIM3 and its ligand (CEACAM1) leads to enhanced antitumor immune responses, im-
proving CRC clearance [127]. Clinical trials are evaluating TSR-022 (anti TIM3 antibody) in
solid tumors, including colorectal cancer (phase 1 NCT02817633). Mouse models demon-
strated that combining LAG3 and PD1 inhibitors overcame tumor resistance to either agent
alone [128]. Similarly, the evaluation of nivolumab with relatlimab (LAG3 monoclonal
antibody) in MSS colorectal cancer patients is underway (phase 2 NCT03642067) (Table 1).
After a combination of favezelimab (Anti-LAG3) and pembrolizumab demonstrated safety
and robust efficacy in the PD-L1 combined positive score (CPS) ≥ 1 MSS mCRC, a phase
3 trial is comparing it to SOC in PD-L1 positive CRC (NCT05064059) (Table 1) [129].

3.6. CEA/CEACAM5
Carcinoembryonic antigen (CEA), a glycoprotein and tumor marker encoded by CEA-
CAM5, is elevated in patients with CRC. Preoperative serum CEA (s-CEA) of > 5 ng/mL
confer worse OS and cancer-specific survival [127,128]. In addition to s-CEA, tissue CEA
(t-CEA) can be assessed using IHC with expression patterns described as apicoluminal
Cancers 2024, 16, 2796 12 of 25

(AL) or diffuse-cytoplasmic (DC). DC pattern t-CEA is associated with a higher rate of


recurrence along with a lower OS and disease-free survival (DFS) relative to the AL pat-
tern [129–131]. Additionally, increased high-intensity t-CEA is associated with a decreased
DFS [131]. The elevated expression of CEACAM5 in colorectal cancer relative to normal
tissue highlights its utility as an antibody-drug conjugate (ADC) target. Early data for
M9140 (Anti-CEACAM5 ADC with a topoisomerase 1 inhibitor) demonstrated a suitable
safety profile and disease control rate (52.5%) in heavily pretreated (≥2 prior lines of ther-
apy) mCRC (NCT05464030) (Table 1) [132]. Cibisatamab (CEACAM5/CD3 T cell–bispecific
antibody) is being tested with atezolizumab in mCRC (NCT03866239) (Table 1).

3.7. c-MET
Mesenchymal–epithelial transition factor (c-MET) is a receptor tyrosine kinase encoded
by MET, responsible for cellular proliferation, angiogenesis, and epithelial–mesenchymal
transition (EMT) [130]. Several studies, along with a meta-analysis, noted that an increased
c-MET expression predicts worse OS and DFS [131,132]. MET amplification correlates
with acquired resistance to anti-EGFR therapy in CRC without KRAS mutations [133].
Combining cetuximab with MET inhibitors suppressed MET-induced anti-EGFR resistance
in CRC cell lines [134]. Early data on ABBV-400, a c-MET-targeted ADC linked with
telisotuzumab (topoisomerase 1 inhibitor), demonstrated a robust safety profile and ORR
(61.9%) in CRC (NCT05029882, NCT06107413) (Table 1) [135].

3.8. ARID1A
ARID1A is a subunit of the SWI/SNF complex, which plays a role in regulating
DNA repair. A loss of function of the SWI/SNF complex promotes genomic instability
and tumor progression. In CRC, ARID1A is a known driver gene, and mutations are
associated with right-sided tumors, MSI-H/dMMR, and BRAF mutations [136]. Studies
suggest that ARID1A mutations may be implicated in both intrinsic and acquired resistance
to cetuximab [137]. Patients enrolled in CALGB/SWOG 80405, a randomized phase III
study evaluating the efficacy of chemotherapy plus cetuximab vs. chemotherapy plus
bevacizumab in first-line CRC, who had ARID1A mutations at baseline, had poorer clinical
outcomes with cetuximab compared to bevacizumab. These findings were supported
in patient-derived xenograft models of extended RAS/BRAF wildtype tumors. In post-
treatment cell-free DNA (cfDNA) analysis of these patients, KRAS and ARID1A mutations
were enriched in those who were treated with cetuximab but not bevacizumab. Additional
CRC genomic analysis suggests that ARID1A and EGFR-pathway mutations are mutually
exclusive, consistent with findings in lung cancer. Further exploration is warranted to
delineate if ARID1A mutations identify a group of patients who benefit from MAPK
inhibition, in addition to being a predictive biomarker of a poor response to cetuximab
treatment. Phase 2 trials are evaluating tislelizumab (PD-1 Antibody) plus fruquintinib
(VEGFR 1/2/3 Inhibitor) for ARID1A-mutated MSS mCRC (NCT05690035) (Table 1).

3.9. PLK1
Polo-like Kinase 1 (PLK1) is a serine/threonine protein kinase family that plays a
role in cell cycle progression by regulating progression to mitosis at the G2/M check-
point [138]. It also plays a role in DNA damage response and cell death pathways. PLK1 is
overexpressed in many tumor types and associated with poor prognosis, making it a
promising therapeutic target. Higher levels of PLK-1/phosphorylated-PLK1 were found
in relapsed/mCRC tissues compared to matched primary CRC tissues, suggesting that
it is a marker of poor prognosis and resistance to oxaliplatin-based chemotherapy [139].
Inhibition of PLK1 in CRC cell lines demonstrated that PLK1 inhibitors may attenuate
oxaliplatin resistance [139]. A Phase 1b/2 trial studied onvansertib, an oral highly selective
PLK1 inhibitor, in combination with FOLFIRI and bevacizumab as a second-line treat-
ment for KRAS-mutated mCRC and found that the combination was well-tolerated with
durable responses [140]. A Phase 2 trial is underway to assess its utility combined with
Cancers 2024, 16, 2796 13 of 25

chemotherapy in the first-line setting for KRAS- or RAS-mutated mCRC (NCT06106308)


(Table 1).

4. The Role of Liquid Biopsy/ctDNA


Liquid biopsies (circulating tumor DNA [ctDNA]) are valuable tools in the manage-
ment of CRC. When a tumor cell dies, or through active secretion, ctDNA is released
in body fluids, most commonly blood [141]. This allows for the detection of mutations,
chromosomal rearrangements, methylation patterns, and different fragment lengths [142].
Liquid biopsies can be performed serially during a patient’s disease course and used to
interpret tumor biology and assess tumor response to therapy [143]. Although tissue biopsy
is the gold standard for molecular profiling, it has several limitations, including insufficient
tissue sampling and a longer turnaround time. A prospective study from Japan noted
ctDNA to have a lower sequencing failure rate (0.1% vs. 10.6%) and a shorter screening time
(11 vs. 33 days, sample acquisition + test duration), ultimately leading to a higher clinical
trial enrollment (9.5% vs. 4.1%) [144]. Tumor heterogeneity and evolution have necessitated
resampling with liquid biopsy mutations that confer resistance to treatments such as RAS
mutations in those receiving anti-EGFR therapy [145]. The concordance of tissue and liquid
biopsy is acceptable, ranging from 77–96%, with NGS-based methods [145].
ctDNA has shown significant promise in detecting minimal residual disease (MRD)
and monitoring for early recurrence in CRC. A prospective study of postsurgical stage III
CRC highlighted that MRD positivity following adjuvant chemotherapy is significantly
associated with an increased risk of recurrence (HR, 7.5; 95% CI, 3.5–16.1; p < 0.001) [146].
These findings were replicated in several studies, even demonstrating prognostic value
with ctDNA positivity having worse OS [147,148]. The prognostic utility of ctDNA has
been leveraged to select CRC patients likely to benefit from adjuvant chemotherapy (ACT),
reducing unnecessary toxicities. Stage II/III CRC patients were prospectively randomized
to SOC or a ctDNA-guided protocol for ACT and demonstrated noninferior DFS and
OS [149]. When evaluating metastatic gastrointestinal cancers, a four week decrease
following systemic therapy initiation predicted the clinical benefit and PFS [149]. Several
trials are utilizing dynamic changes in ctDNA to guide treatment decisions in mCRC
(NCT04786600, NCT03844620, NCT05062317). Liquid biopsy lacks sensitivity in detecting
fusions and copy number losses while having a higher TMB relative to tissue biopsy [150].
The utility of ctDNA has broadened from its initial use in the advanced/metastatic setting
for molecular profiling and detection of acquired resistance mechanisms to the identification
of MRD and early detection.

5. Future Directions
NGS has altered the paradigm of genomics research, offering unparalleled capabilities
for analyzing DNA and RNA molecules in a high-throughput and cost-effective manner.
Third-generation sequencing has allowed for longer read lengths, real-time sequencing,
minimal sample preparation, and direct detection of epigenetic modifications, providing
a comprehensive understanding of genomic structure [151]. Using NGS in ctDNA offers
a minimally invasive approach to monitor disease progression and treatment response.
Studying the dynamics of these biomarkers over time and across different treatment
regimens can provide valuable insights into the tumor landscape and inform personalized
treatment strategies [152].
CRC is known to exhibit a high intratumor heterogeneity, with different subpopula-
tions of tumor cells harboring distinct genetic and transcriptomic profiles. Single-cell RNA
sequencing (scRNA-seq) evaluates the heterogeneity at the single-cell level, enabling the
identification of rare cell subpopulations that may play crucial roles in disease progression,
metastasis, and treatment resistance [153]. Additionally, scRNA-seq can delineate the com-
plex interactions between tumor cells and immune cells, potentially identifying biomarkers
related to immunosuppression or immune evasion.
Cancers 2024, 16, 2796 14 of 25

Multiomics integrates genomic, transcriptomic, proteomic, and epigenomic data along


with clinical metadata through artificial intelligence (AI) and bioinformatics tools. By
combining data from various omic levels, researchers can gain a more comprehensive
understanding of the underlying mechanisms, regulatory pathways, and functional rela-
tionships with CRC. Utilizing feature selection and extraction techniques such as principal
component analysis, researchers can reduce the dimensionality of multiomics data while
preserving the variance and information [154]. Machine learning (ML) models have demon-
strated a remarkable accuracy in predicting responses to first-line chemotherapy based on
multiomics data [155].
With advances in molecular testing and modeling, we highlight emerging evidence on
novel pathways and biomarkers that may double as therapeutic targets. HLAs are divided
into two classes, HLA class I and class II, which present peptides to CD8+ T cells and
CD4+ T cells, respectively. Reduced expression of HLA class I genes has been associated
with immune evasion in immunotherapy-naive MSI-H/dMMR CRC, suggesting a possible
explanation for primary resistance to ICI [156,157]. HLA class I and II SNPs predict
outcomes in first-line treatment in mCRC, with differential effects based on biologic agent
and chemotherapy backbone [158,159]. While not prognostic, MCL-1, an anti-apoptotic
protein from the BCL-2 family, induces chemoresistance and plays a crucial role in CRC
by maintaining intestinal homeostasis [160–162]. Given that MCL-1 and BCL-xL are co-
expressed in late-stage CRC, this chemoresistance can be countered by coadministration
of A-1331852 (BCL-xL inhibitor) and A-1210477 (MCL-1 inhibitor) [162]. A high MCL-
1 expression also correlates with an increased M1 and M2 macrophage infiltration in CRC
tumors, affecting cancer cell proliferation and EMT [163,164].
The circadian rhythm, regulated by the core clock genes (BMAL1 and CLOCK), is an-
other pathway closely linked with CRC proliferation and metastasis [165,166]. BMAL1 sup-
presses a hippo-dependent, self-renewal pathway in the intestinal epithelium such that
the loss of BMAL1 in knockout mouse models results in the upregulation of stem cell
signaling and increased CRC growth [167]. BMAL1 drives the transcription of VEGFA
conferring resistance to bevacizumab treatment, therefore combining bevacizumab with
a CRY2 stabilizer (SHP1705) to inhibit BMAL1 activity led to a decreased tumor volume
and an improved OS in mouse models [168]. Like BMAL1, dihydropyrimidine dehy-
drogenase (DPD) is another protein involved in therapeutic resistance. A high tumoral
DPD expression is associated with 5-FU resistance in CRC; consequently, combining 5-FU
with DPD inhibitors (S1PR2 inhibitors or SLR080811) improves efficacy [169–171]. DPD
expression positively correlates with CD4+ T cells, CD8+ T cells, and macrophages in the
TME, in addition to the expression of immune checkpoint markers (PD-L1 and PD-1) [172].
This suggests that DPD not only has a predictive value for 5-FU resistance but also holds
potential for immunotherapy-treated CRC.
Both neurotransmitters and ferroptosis play a role in modulating the immune re-
sponse and are growing areas of CRC research. Neurotransmitters in gastrointestinal
tumors facilitate interactions between cancer and immune cells, promoting oncogenic
processes [173–176]. The expression of neurotransmitter receptors on immune cells affects
tumor immune responses and may influence immune-targeting treatments [174]. Block-
ing receptors like muscarinic receptor 3 (M3R) and monoamine oxidase A (MAOA) have
shown decreased tumor growth and altered immune responses in CRC models [177,178].
These findings suggest that neurotransmitters have potential as predictive biomarkers and
may pave the way for novel therapies targeting neurotransmitters to overcome therapy
resistance [179]. Ferroptosis, an iron-dependent form of cell death, is regulated by genes
involved in iron metabolism and has been implicated in CRC tumor biology [180–182].
In CRC, the altered expression of ferroptosis-related genes predicts OS and the immune
response [183–187]. Ferroptosis is being explored in combination therapy with ICIs as
a promising treatment strategy to increase sensitivity and overcome resistance [188,189].
Glutamine metabolism regulates ferroptosis by contributing to the synthesis of glutathione
(GSH) and through the process of glutaminolysis [190]. Oncogenic KRAS reprograms
Cancers 2024, 16, 2796 15 of 25

cellular metabolism, limiting these processes and thereby making KRAS-mutant tumors
more susceptible to ferroptosis induction as a potential clinical strategy [191].
As the era of targeted therapies continues to evolve, the development of companion
diagnostics and predictive biomarkers becomes increasingly crucial. Identifying biomarkers
that can accurately predict the response to targeted agents will maximize therapeutic
efficacy while minimizing unnecessary toxicities and healthcare costs.

6. Conclusions
The predictive capacity of MSI-H, RAS, and BRAF has necessitated a molecular-
targeted approach in CRC. The MSI status confers substantial improvements in clinical
outcomes and decreased toxicities through ICI, offering patients long-term survival or near
curative results [69]. BRAF V600E had a negative prognostic value but is now predictive
of the response to combined targeted BRAF/EGFR inhibition, doubling survival over
standard treatments [51]. While the prevalence of these alterations constitutes the minority
of CRCs, they underscore the necessity of identifying predictive biomarkers.

Author Contributions: All authors have made substantial contributions to the review. Conceptual-
ization: K.A., A.W., H.-J.L. and F.B.; writing, review, and/or revision of the manuscript: all authors;
study supervision/guarantor: K.A., A.W., H.-J.L. and F.B. All authors have read and agreed to the
published version of the manuscript.
Funding: This study was partly supported by the National Cancer Institute (grant number P30CA014089)
[to H.-J.L.], the Gloria Borges WunderGlo Foundation, Dhont Family Foundation, Victoria and Philip
Wilson Research Fund, San Pedro Peninsula Cancer Guild, Ming Hsieh Research award, and Daniel
Butler Research Fund. The content is solely the responsibility of the authors and does not necessarily
represent the official views of the National Institutes of Health.
Conflicts of Interest: H.-J.L. reports receiving honoraria from consultant/advisory board membership
for Merck Serono, Bayer, and Genentech. The remaining authors declare that the research was
conducted in the absence of any commercial or financial relationships that could be construed as a
potential conflict of interest.

Abbreviations

5-FU 5-fluorouracil
ADC antibody-drug conjugate
AI artificial intelligence
AL apicoluminal
BRAFV600E BRAF mutation with valine substitution for glutamic acid at amino acid 600
c-MET mesenchymal–epithelial transition factor
CCR5 C-C motif chemokine receptor 5
CEA carcinoembryonic antigen
cfDNA cell-free DNA
CMS consensus molecular subtype
CRC colorectal cancer
ctDNA circulating tumor DNA
CXCR4 CXC chemokine receptor 4
DC diffuse-cytoplasmic
DFS disease-free survival
DPD dihydropyrimidine dehydrogenase
EGFR epidermal growth factor receptor
EMT epithelia–mesenchymal transition
FDA Food and Drug Administration
FISH fluorescence in situ hybridization
GSH glutathione
HLA human leukocyte antigen
Cancers 2024, 16, 2796 16 of 25

ICI immune checkpoint inhibitor


IHC immunohistochemistry
LAG3 lymphocyte-activation gene 3
M3R muscarinic receptor 3
MAOA monoamine oxidase A
Mb megabase
mCRC metastatic colorectal cancer
MHC major histocompatibility complex
ML machine learning
MRD minimal residual disease
MSI-H/dMMR microsatellite instability or mismatch repair deficiency
MSS microsatellite stable
NCCN National Comprehensive Cancer Network
NGS next-generation sequencing
NR not reached
NSCLC non-small cell lung cancer
OS overall survival
PFS progression-free survival
PI3K phosphatidylinositol 3-kinase
PLK1 polo-like Kinase 1
s-CEA serum carcinoembryonic antigen
scRNA-seq single-cell RNA sequencing
SNP single nucleotide polymorphism
SOC standard of care
t-CEA tissue carcinoembryonic antigen
TAMs tumor-associated macrophages
TCR T cell receptor
TILs tumor infiltrating lymphocytes
TIM3 T cell immunoglobulin and mucin domain-containing protein 3
TKI tyrosine kinase inhibitor
TMB tumor mutational burden
TME tumor microenvironment
Treg regulatory T cell
VEGF-A vascular endothelial growth factor A
WES whole exome sequencing

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